Novel Affibody Molecules Specifically Bind to SARS-CoV-2 Spike Protein and Efficiently Neutralize Delta and Omicron Variants

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented public health disaster in human history, and its spike (S) protein is the major target for vaccines and antiviral drug development. Although widespread vaccination has been well established, the viral gene is prone to rapid mutation, resulting in multiple global spread waves. Therefore, specific antivirals are needed urgently, especially those against variants. In this study, the domain of the receptor binding motif (RBM) and fusion peptide (FP) (amino acids [aa] 436 to 829; denoted RBMFP) of the SARS-CoV-2 S protein was expressed as a recombinant RBMFP protein in Escherichia coli and identified as being immunogenic and antigenically active. Then, the RBMFP proteins were used for phage display to screen the novel affibody. After prokaryotic expression and selection, four novel affibody molecules (Z14, Z149, Z171, and Z327) were obtained. Through surface plasmon resonance (SPR) and pseudovirus neutralization assay, we showed that affibody molecules specifically bind to the RBMFP protein with high affinity and neutralize against SARS-CoV-2 pseudovirus infection. Especially, Z14 and Z171 displayed strong neutralizing activities against Delta and Omicron variants. Molecular docking predicted that affibody molecule interaction sites with RBM overlapped with ACE2. Thus, the novel affibody molecules could be further developed as specific neutralization agents against SARS-CoV-2 variants. IMPORTANCE SARS-CoV-2 and its variants are threatening the whole world. Although a full dose of vaccine injection showed great preventive effects and monoclonal antibody reagents have also been used for a specific treatment, the global pandemic persists. So, developing new vaccines and specific agents are needed urgently. In this work, we expressed the recombinant RBMFP protein as an antigen, identified its antigenicity, and used it as an antigen for affibody phage-display selection. After the prokaryotic expression, the specific affibody molecules were obtained and tested for pseudovirus neutralization. Results showed that the serum antibody induced by RBMFP neutralized Omicron variants. The screened affibody molecules specifically bound the RBMFP of SARS-CoV-2 with high affinity and neutralized the Delta and Omicron pseudovirus in vitro. So, the RBMFP induced serum provides neutralizing effects against pseudovirus in vitro, and the affibodies have the potential to be developed into specific prophylactic agents for SARS-CoV-2 and its variants.

6. Common English language errors, for example, Fig. 2 and 3 legends. Overall, the text is good. Just some minor corrections are needed in the manuscript.
Reviewer #2 (Comments for the Author): Overview: This manuscript provides an interesting development narrative of four novel affibodies targeting the RBMFP of the SARS-CoV-2 spike protein. Effort was taken to evaluate the antigenicity of the RBMFP protein and understand whether affibodies produced by a phage display platform are capable of neutralizing SARS-CoV-2 variants. Although there is a data to support some of the conclusions made in this manuscript, this reviewer would like to address several challenges: Major comments: The neutralization data in this study are not well supported. Single fluorescent images are provided and neutralization extrapolated without an understanding of cell density in each image or appropriate controls in some cases (see detailed comments). Without this information it is extremely difficult to validate the presence of true neutralization and the major emphasis of this manuscript. This reviewer would appreciate additional evidence of affibody binding to endogenous respiratory cell lines that express human ACE2 to ensure that binding remains in an unmodified / biologically relevant cell line. Detailed comments: Single references of critical statements are inadequate to support many statements that can be validated by multiple studies at this point in the pandemic. There is very little methodological description of the serological ELISA that was performed and would not be reproducible from this method. This should be described in much better detail. Figure 4C: Statistical analysis of titer, area under the curve, EC50 etc. would help provide additional support for this conclusion. Figure 4D-E: Serum neutralization should be validated with quantification of fluorescent intensity or by additional surrogate assays. Single images demonstrate a potential trend however this does not adequately support the conclusion that all RBMFP+ sera provides a neutralizing effect. In addition, the PBS group is not represented in either Figure 4D and 4E which does not provide a point of comparison and no phase contrast images providing a reference for cell number per image is provided. Figure 5C-D: Labeling of the gels and blots would help provide additional clarity to the figure. Figure 7: Phase contrast images should be provided and luminescent / fluorescent intensity values should be normalized to the area of cells per well. Without normalizing to cell number / area per image it is possible that a reduction in luminescent / fluorescent intensity is merely an artifact of fewer cells per image. Additionally, a statement on the number of images analyzed per sample should be provided. Figure 7D: Statistical analysis of multiple groups should be performed using ANOVA, not t-test. Lines 56-57: There is no evidence provided to suggest that the RBMFP provides neutralizing protection in vivo in this manuscript Line 69: Reference number seven only refers to vaccine resistance of the delta variant. This statement should be qualified by additional studies demonstrating viral resistance to existing vaccines. Lines 61-71: Statements in introduction should be referenced in more detail. Many studies exist supporting this logic and single references do not adequately describe the literature encompassing this topic. Lines 73-79: References should be included to support these statements. Lines 99-101: Further references regarding the structure, function, and utility of affibodies would be beneficial. For example: Frejd, F., Kim Line 178: What does "correct sequence" mean? Line 225: Reference the mechanism of SARS-CoV-2 entry to cells. Line 227: "High level of specific...". This phrasing is not valid. No comparison for whether this degree of IgG production qualifies as high. Please revise by comparing to a control or remove "high level". Lines 253-254: This reviewer would appreciate clarity from the author's as to whether there is evidence an advantage of affibodies against heavily glycosylated proteins over mAbs. If there is no evidence to support this, this statement should be revised to reflect this. Lines 247, 257-262: The KD of bamlanivimab is 0.071nM and blocks ACE2 spike interactions with an IC50 of 0.17nM. The KD of estevimab is 6.45nM and blocks ACE2-spike interactions with an IC50 of 0.32nM. It would be useful to put the affinity and neutralizing capacity of the affibodies in direct context with these existing FDA-authorized drugs for discussion. The values provided in line 247 are misleading if not in the same units as the affibody molecules. KD and IC50 values of affibody molecules greater than 10-fold higher than existing drugs should be discussed and statements regarding prospective efficacy tempered accordingly.

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Overview: This manuscript provides an interesting development narrative of four novel affibodies targeting the RBMFP of the SARS-CoV-2 spike protein. Effort was taken to evaluate the antigenicity of the RBMFP protein and understand whether affibodies produced by a phage display platform are capable of neutralizing SARS-CoV-2 variants. Although there is a data to support some of the conclusions made in this manuscript, this reviewer would like to address several challenges:

Major comments:
The neutralization data in this study are not well supported. Single fluorescent images are provided and neutralization extrapolated without an understanding of cell density in each image or appropriate controls in some cases (see detailed comments). Without this information it is extremely difficult to validate the presence of true neutralization and the major emphasis of this manuscript.
This reviewer would appreciate additional evidence of affibody binding to endogenous respiratory cell lines that express human ACE2 to ensure that binding remains in an unmodified / biologically relevant cell line.

Detailed comments:
Single references of critical statements are inadequate to support many statements that can be validated by multiple studies at this point in the pandemic.
There is very little methodological description of the serological ELISA that was performed and would not be reproducible from this method. This should be described in much better detail. Figure 4C: Statistical analysis of titer, area under the curve, EC 50 etc. would help provide additional support for this conclusion. Figure 4D-E: Serum neutralization should be validated with quantification of fluorescent intensity or by additional surrogate assays. Single images demonstrate a potential trend however this does not adequately support the conclusion that all RBMFP + sera provides a neutralizing effect. In addition, the PBS group is not represented in either Figure 4D and 4E which does not provide a point of comparison and no phase contrast images providing a reference for cell number per image is provided.   Line 178: What does "correct sequence" mean?
Line 225: Reference the mechanism of SARS-CoV-2 entry to cells. Line 227: "High level of specific…". This phrasing is not valid. No comparison for whether this degree of IgG production qualifies as high. Please revise by comparing to a control or remove "high level".
Lines 253-254: This reviewer would appreciate clarity from the author's as to whether there is evidence an advantage of affibodies against heavily glycosylated proteins over mAbs. If there is no evidence to support this, this statement should be revised to reflect this.
Lines 247, 257-262: The K D of bamlanivimab is 0.071nM and blocks ACE2 spike interactions with an IC 50 of 0.17nM. The K D of estevimab is 6.45nM and blocks ACE2-spike interactions with an IC 50 of 0.32nM. It would be useful to put the affinity and neutralizing capacity of the affibodies in direct context with these existing FDA-authorized drugs for discussion. The values provided in line 247 are misleading if not in the same units as the affibody molecules. K D and IC 50 values of affibody molecules greater than 10-fold higher than existing drugs should be discussed and statements regarding prospective efficacy tempered accordingly.

Response to reviewers' comments
Reviewer #1 (Comments for the Author): The authors selected the domains of RBM and FP from SARS-CoV-2 S protein that binds and fuses with cell membrane receptors as targets, prepared the RBMFP fusion protein using a prokaryotic expression system, and identified its immunogenicity and antigenicity. Further, they selected the novel affibodies with phage-display technology and obtained affibodies with binding affinity to RBMFP. Then, the neutralization effect of affibodies on SARS-CoV-2 pseudovirus was evaluated.
The authors present compelling data to support the binding specificity of these affibodies and their neutralizing efficacy against SARS-CoV-2 pseudovirus. The paper is well written and interesting, especially Z14 and Z171 neutralize against Omicron variants, a dominant epidemic today, so the researchers have potential and application prospects; however, some concerns have been raised during the revision process

Response to Reviewer 1:
Here is a point-by-point response to the reviewer's comments and concerns. and only the RBD within the S1 subunit is well-exposed to antibody recognition (4), thus modulating the variants' escape from immune surveillance that induced by vaccines developed. In contrast, RBMFP is glycosylation free but still has RBM and other major antigen regions, such as FP and cleavage sites, which also neutralize some variants. Further, most full-length S protein vaccines and completely inactivated vaccines are prepared in eukaryotic expression systems. While the RBMFP are prepared in the prokaryotic expression system and may have advantages in future use (5).

Q:
The authors point out that they have produced "high-affinity" binders.
The authors report single-digit micromolar KDs calculated based on the binding kinetics using SPR. Although there is such clear cell binding and measurable in vitro, a binder with μM affinity may not be ideal for the preventive or therapeutic agent. Whether further improvements are needed, or how to modify them further to increase their affinity for further development and application?
A: We understand the reviewer's concerns. Here, we used an SPR assay that confirmed 4 binders with nM~μM level affinity. The binding affinity is lower than some mAbs (6). Other researchers reported that further modifications into the dimer or trimer of these affibodies could enhance their binding affinity for their target (7 A: Thank you for the comment. As shown in Fig.8 and Table 2  Q: Common English language errors, for example, Fig. 2 and 3 legends.
Overall, the text is good. Just some minor corrections are needed in the manuscript.

A:
We agree with the reviewer's assessment, and changes have been made accordingly in the revised manuscripts ( Fig. 2 and 3 legends).

Reviewer #2
Overview: This manuscript provides an interesting development narrative of four novel affibodies targeting the RBMFP of the SARS-CoV-2 spike protein. Effort was taken to evaluate the antigenicity of the RBMFP protein and understand whether affibodies produced by a phage display platform are capable of neutralizing SARS-CoV-2 variants. Although there is a data to support some of the conclusions made in this manuscript, this reviewer would like to address several challenges:

Response to Review2:
We feel great thanks for your professional review work on our article. As you are concerned, several challenges need to be addressed. According to your suggestions, we have made extensive corrections to our previous draft, and the detailed corrections are listed below.  Figure   4D, E); the cell numbers were counted through Image Pro Plus 6.0 software and, also added ( Fig. S2 B).  Q: In addition, the PBS group is not represented in either Figure 4D and 4E which does not provide a point of comparison and no phase contrast images providing a reference for cell number per image is provided.
A: Thank you for the comments. In this study, a culture medium was used to dilute mouse serum, and no mice serum added group was set as a negative control, which we represented as serum-free in the original manuscript. Furthermore, we also provided white light images in the re-